Large numbers of blood cells are required for the treatment of blood-related diseases or surgical treatments. Among blood cells, a platelet which is a cell indispensable for blood coagulation and hemostasis is one of particularly important blood cells. The platelet is in high demand in leukemia, bone marrow transplantation, anticancer treatment, and the like so that necessity of stable supply of it is high. The platelet has so far been supplied stably by, as well as a method of collecting it from the blood donated by donors, a method of administering a drug having a TPO-like (mimetic) structure, a method of differentiating megakaryocytes from the cord blood or bone marrow cells, or the like method. Recently, there has been developed a technology of inducing in vitro differentiation of pluripotent stem cells such as ES cells or iPS cells to prepare blood cells such as platelets.
The present inventors have established a technology of inducing differentiation of megakaryocytes and platelets from human ES cells and shown effectiveness of ES cells as a source of platelets (Patent Document 1 and Non-patent Document 1). In addition, the present inventors have established a method for preparing megakaryocytes and platelets from iPS cells and have enabled dissolution of the problem of compatibility of a human leukocyte antigen (HLA) which inevitably occurred in transfusion of platelets derived from ES cells (Patent Document 2).
Further, with a view to overcoming the problem of the amount of platelets and the like prepared from stem cells, the present inventors have found a method of establishing and thereby preparing an immortalized megakaryocyte progenitor cell line from the stem cells and thus, have developed an important technology for in vitro preparation of a large amount of platelets and the like (Patent Document 3).
In vivo, megakaryocytes form pseudopodial formation called proplatelets (platelet progenitors), fractionate their cytoplasm, and release platelets. Polyploidization of megakaryocytes is thought to occur by endomitosis until they release platelets. Endomitosis of megakaryocytes is multipolar mitosis not accompanied with cleavage furrow formation and spindle extension and caused by abnormal karyokinesis and cytoplasm mitosis. As a result of endomitosis, cells containing several segmented nuclei are formed. Polyploidization of megakaryocytes is induced by repetition of such endomitosis.
Many study results have been reported to date on polyploidization of megakaryocytes. Lodier, et al. have elucidated (Non-patent Document 1) that in endomitosis of megakaryocytes, localization of nonmuscle cell myosin II in a contractile ring has not been recognized in spite of formation of cleavage furrow and defects occur in contractile ring formation and spindle extension. It has been shown that such abnormalities in contractile ring or spindle extension become more marked by inhibiting RhoA and Rock activities (Non-patent Document 2). RhoA accumulates at the cleavage furrow and promotes activation of some effector factors including Rho kinase (Rock), citron kinase, LIM kinase, and mDia/formins. These results suggest that by inhibiting the activities of factors such as RhoA and Rock involved in formation of a contractile ring, endomitosis of megakaryocytes is promoted. There is also a report that when a Rho signal positioned downstream of integrin alpha2/beta1 is reinforced, formation of proplatelets of immature megakaryocytes before polyploidization is inhibited.
It is reported that all trans retinoic acid (ATRA), a transcription factor and valproic acid which is known as a histone deacetylase inhibitor are involved in differentiation of megakaryocytes. Schweinfurth, et al. have found that treatment of immature megakaryocytes with all trans retinoic acid or valproic acid promotes polyploidization (Non-patent Document 3). Further, it is reported that polyploidization of megakaryocytes is promoted when p53, a cancer suppressor gene product is knocked down (Non-patent Document 4).
It has also been shown that as an influence on a differentiation procedure of megakaryocytes, culturing immature megakaryocytes at 39° C., temperature higher than conventional culturing temperature, promotes induction to polyploidized mature megakaryocytes and formation of proplatelets (Non-patent Document 5).